Linear regression was used to estimate the amount of virus still present in these patients with viral loads too low for detection at the time of withdrawing from therapy. Assuming 3000ml plasma per body, the median estimated residual total body plasma virus was 4.6log/body plasma (or approximately 10 HIV RNA copies/ml) with a range 0.7-5.3log (Table 2). In all but one patient, the lower limit of the 95% confidence interval was above zero. The extrapolation may not be reliable for this patient (number 4), as only three data points were available, resulting in an extremely wide confidence interval. This patient also died shortly after day 21. The baseline viral load of the other five patients correlated with the estimated amount of residual HIV (r2=0.88; P=0.02) and inversely with the time required to rebound to 500copies/ml (Pearson r2=0.85; P=0.03), suggesting that the net reduction in viral load achieved was fairly similar in all five patients. The estimated decrease in total body plasma HIV RNA during HAART therapy was 3.0 to 4.7log (excluding patient 4).
There was no obvious correlation between the rate of viral rebound and the baseline plasma viral load (r2=0.01), the baseline CD4 cell count (r2=0.5) or the duration of time patients had remained below 500 HIV RNA copies/ml (r2=0.01).
Total CD4 cell counts and helper:suppressor cell ratios were monitored for one individual (Patient 6) during the 21-day viral-rebound period and no apparent changes were observed during this time. In another individual (Patient 4), the change in peripheral blood HIV DNA in blood mononuclear cells was measured using a quantitative HIV DNA polymerase chain reaction (PCR) assay. HIV DNA levels increased approximately twofold in the 21 days following withdrawal from therapy (data not shown).
The rapid rates of viral rebound observed in these six patients clearly show that complete viral clearance had not occurred despite up to 18 months of viral suppression below 500copies/ml HIV RNA. As little as 1 week of withdrawal from HAART therapy resulted in plasma HIV RNA levels of greater than 500copies/ml, emphasizing the requirement for strict adherence to these drug regimens if HIV replication is to be minimized. This rapid viral rebound should be considered when interpreting sudden fluctuations in HIV RNA levels in clinical practice, as well as in clinical trials where the occurrence of ‚detectable‚ virus is an endpoint . Individuals should be very closely monitored between 7 and 10 days after stopping therapy to test for the possibility of HIV eradication.
The regression analysis estimates presented here suggest that thousands of copies of plasma HIV RNA may be present in the body at a time when even the most sensitive quantitative PCR assays yield undetectable results. Given the practical limits on sample size, only about 0.1% of the total body plasma can reasonably be analyzed; consequently, even the newest generation HIV RNA assays will not be sufficiently sensitive to demonstrate that the plasma (let alone the rest of the body) is free of HIV RNA. Other approaches for the routine monitoring of HIV infection in patients receiving HAART are, therefore, required.
There is surprisingly little information on the rebound of viral load upon stopping therapy after a sustained reduction in plasma viral load. Patients discontinuing 8-19 days of nucleoside analogue therapy  or longer periods of triple combination therapy  were reported to have a transient rise in plasma viral load above baseline. We previously reported a case of a patient with HIV RNA levels consistently below 50copies/ml for 28 months whose plasma viral load rebounded to levels far greater than that observed at baseline within 1 month of stopping therapy . Of interest, there is also one report of a complete absence of viral rebound in two patients for up to 1 year after withdrawing from combination therapy including didanosine and hydroxyurea .
The data presented here provide no evidence that the amount of residual plasma virus is decreasing with increasing time on therapy, as expected if viral eradication were to occur as a result of HAART. Latently infected or slowly cleared reservoirs of HIV [7-9] and/or residual HIV replication, particularly in tissue compartments other than plasma , could account for the lack of total viral clearance. It is feasible that current HAART simply is not sufficiently potent to inhibit viral replication completely and results in a new quasi-equilibrium of plasma viral load that is 3-5log lower than pre-therapy levels. This is consistent with evidence of viral evolution in both the plasma HIV RNA and DNA envelope sequences (though no evidence of evolution of drug resistance) in a patient over a 28-month period during which HAART had reduced plasma viral load below 50copies/ml  unpublished data].
The rates of viral load increase observed here are only slightly slower than the ≊0.5log/day reported during primary infection  or early interruption of triple combination therapy , and they are very similar to those observed for rebound of a resistant variant (V82A) selected during ritonavir monotherapy .
These results are limited by to the small number of patients analyzed. In addition, a limitation of the analyses is that the extrapolation to the moment of stopping therapy assumes a constant rate of viral increase and that this increase began at the time that patients stopped taking medication. Furthermore, patients choosing to withdraw from HAART therapy may not be representative of most patients. Nonetheless, these results suggest that many patients who choose to stop HAART after remaining below 500copies/ml HIV RNA for a considerable period of time will have increases in plasma viral HIV RNA of about 0.2log/day and reach easily detectable levels of HIV RNA within 1 or 2 weeks after stopping therapy.
We thank Cindy Christopherson, Shirley Kwok and John Sninsky at Roche Molecular Systems for performing the HIV DNA measurements.
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